We previously demonstrated that the AT2 receptor is a
glycoprotein containing N-linked
oligosaccharide side chains and that the marked disparity between the sizes of AT2 receptors from different tissues was related to different degrees of N-glycosylation. In the present study, we used an inhibitor of N-glycosylation,
tunicamycin, as well as an
endoglycosidase,
glycopeptidase-F, to examine the contribution of
carbohydrate moieties to the
ligand-binding properties, cell-surface expression and apparent molecular mass of AT2 receptors of rat
pheochromocytoma cells (PC-12 cells). Photoaffinity labelling of cell-surface AT2 receptors revealed that PC-12 cells grown in the presence of
tunicamycin expressed, in addition to the previously described 140 kDa receptor, lower-molecular-mass receptors of 63 kDa, 47 kDa and 32 kDa.
Lectin affinity chromatography revealed that the 63 kDa and the 47 kDa receptors are partially glycosylated and that the 32 kDa receptor is completely deglycosylated. Competitive binding experiments were carried out on
tunicamycin-treated cells that expressed predominantly the 63 kDa or the 47 kDa receptors. Both receptor forms exhibited a high affinity for
angiotensin II, although a slight decrease (of about 2-fold) was consistently observed on
tunicamycin-treated cells as compared with control cells.
Endoglycosidase digestion of AT2 receptors of PC-12 cells also yielded smaller receptor forms of 47 kDa and 32 kDa. Similarly,
angiotensin II showed a high but slightly decreased binding affinity (of about 2-fold) for deglycosylated membranes as compared with control membranes. In conclusion, the stepwise action of
tunicamycin suggests the presence of at least three N-linked
oligosaccharide side chains on the AT2 receptor of PC-12 cells. These
oligosaccharide side chains have a minor contribution to the affinity of the receptor. Interestingly, glycosylation is not an essential requirement for the expression of AT2 receptor at the surface of PC-12 cells.